Estimating Wolf Population Size and Dynamics by Field Monitoring and Demographic Models: Implications for Management and Conservation.

We estimated the current size and dynamics of the wolf population in Tuscany and investigated the trends and demographic drivers of population changes. Estimates were obtained by two different approaches: (i) mixed-technique field monitoring (from 2014 to 2016) that found the minimum observed pack number and estimated population size, and (ii) an individual-based model (run by Vortex software v. 10.3.8.0) with demographic inputs derived from a local intensive study area and historic data on population size. Field monitoring showed a minimum population size of 558 wolves (SE = 12.005) in 2016, with a density of 2.74 individuals/100 km2. The population model described an increasing trend with an average annual rate of increase λ = 1.075 (SE = 0.014), an estimated population size of about 882 individuals (SE = 9.397) in 2016, and a density of 4.29 wolves/100 km2. Previously published estimates of wolf population were as low as 56.2% compared to our field monitoring estimation and 34.6% in comparison to our model estimation. We conducted sensitivity tests to analyze the key parameters driving population changes based on juvenile and adult mortality rates, female breeding success, and litter size. Mortality rates played a major role in determining intrinsic growth rate changes, with adult mortality accounting for 62.5% of the total variance explained by the four parameters. Juvenile mortality was responsible for 35.8% of the variance, while female breeding success and litter size had weak or negligible effects. We concluded that reliable estimates of population abundance and a deeper understanding of the role of different demographic parameters in determining population dynamics are crucial to define and carry out appropriate conservation and management strategies to address human-wildlife conflicts.

The masks of Lorenzo Tenchini: their anatomy and surgical/bioengineering clues.

An academic, anatomist, and Lombrosian psychiatrist active at the University of Parma in Italy at the end of the 19th century, Lorenzo Tenchini produced ceroplastic-like masks that are unique in the anatomical Western context. These were prepared from 1885 to 1893 with the aim of 'cataloguing' the behaviour of prison inmates and psychiatric patients based on their facial surface anatomy. Due to the lack of any reference to the procedure used to prepare the masks, studies were undertaken by our group using X-ray scans, infrared spectroscopy, bioptic sampling, and microscopy analysis of the mask constituents. Results showed that the masks were stratified structures including plaster, cotton gauze/human epidermis, and wax, leading to a fabrication procedure reminiscent of 'additive layer manufacturing'. Differences in the depths of these layers were observed in relation to the facial contours, suggesting an attempt to reproduce, at least partially, the three-dimensional features of the facial soft tissues. We conclude the Tenchini masks are the first historical antecedent of the experimental method for face reconstruction used in the early 2000s to test the feasibility of transferring a complete strip of face and scalp from a deceased donor to a living recipient, in preparation for a complete face transplant. In addition, the layering procedure adopted conceptually mimics that developed only in the late 20th century for computer-aided rapid prototyping, and recently applied to bioengineering with biomaterials for a number of human structures including parts of the skull and face. Finally, the masks are a relevant example of mixed ceroplastic-cutaneous preparations in the history of anatomical research for clinical purposes.

A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid.

Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.

Human adipose stem cells induced to osteogenic differentiation by an innovative collagen/hydroxylapatite hybrid scaffold.

Novel biomaterials are of paramount importance for bone regrowth. In this study, we investigated human adipose stem cells (hASCs) for osteogenic, osteoconductivity, and osteoinductivity effects of an innovative collagen/hydroxylapatite hybrid scaffold. In hASCs that were grown on this scaffold, osteogenic genes were analyzed for their expression profiles, together with adhesion and extracellular matrix genes. In hASC integrins, basement membrane constituents and collagens were up-regulated, together with cell proliferation. In addition, expression of osteopontin and activated focal adhesion kinase was studied at the protein level. Our in vitro data indicate that hASCs, together with hybrid biomaterial, is an important model of study to investigate in vitro bone induction.-Mazzoni, E., D'Agostino, A., Manfrini, M., Maniero, S., Puozzo, A., Bassi, E., Marsico, S., Fortini, C., Trevisiol, L., Patergnani, S., Tognon, M. Human adipose stem cells induced to osteogenic differentiation by an innovative collagen/hydroxylapatite hybrid scaffold.

Visualizing sound: counting wolves by using a spectral view of the chorus howling.

INTRODUCTION: Monitoring large carnivores is a central issue in conservation biology. The wolf (Canis lupus) is the most studied large carnivore in the world. After a massive decline and several local extinctions, mostly due to direct persecutions, wolves are now recolonizing many areas of their historical natural range. One of the main monitoring techniques is the howling survey, which is based on the wolves' tendency to use vocalisations to mark territory ownership in response to howls of unknown individuals. In most cases wolf howling sessions are useful for the localisation of the pack, but they provide only an aural estimation of the chorus size. We tested and present a new bioacoustic approach to estimate chorus size by recording wolves' replies and visualising choruses through spectrograms and spectral envelopes. To test the methodology, we compared: a) the values detected by visual inspections with the true chorus size to test for accuracy; b) the bioacoustic estimations of a sample of free-ranging wolves' replies developed by different operators to test for precision of the method; c) the aural field estimation of chorus size of a sample of free-ranging wolves' replies with the sonogram analysis of the same recordings to test for difference between methods. RESULTS: Visual inspection of the chorus by spectrogram and spectrum proved to be useful in determining the number of concurrent voices in a wolf chorus. Estimations of chorus size were highly correlated with the number of wolves counted in a pack, and 92 % of 29 known chorus sizes were recognized by means of bioacoustic analysis. On the basis of spectrographic evidence, it was also possible to identify up to seven concurrent vocalisations in a chorus of nine wolves. Spectral analysis of 37 free ranging wolves' replies showed a high correlation between the chorus size estimations of the different operators (92.8 %), but a low correlation with the aural estimation (59.2 %). CONCLUSIONS: Wolf howling monitoring technique could be improved by recording wolves' replies and by using bioacoustic tools such as spectrograms and spectral envelopes to determine the size of the wolf chorus. Compared with other monitoring techniques (i.e., genetic analysis), bioacoustic analysis requires widely available informatic tools (i.e., sound recording set of devices and sound analysis software) and a low budget. Information obtained by means of chorus analysis can also be combined with that provided by other techniques. Moreover, howls can be recorded and stored in audio file format with a good resolution (i.e. in "Wave" format), thus representing a useful tool for future listening and investigations, which can be countlessly employed without risks of time deterioration.

Predicting the Spatial Distribution of Wolf (Canis lupus) Breeding Areas in a Mountainous Region of Central Italy.

Wolves (Canis lupus) in Italy represent a relict west European population. They are classified as vulnerable by IUCN, though have increased in number and expanded their range in recent decades. Here we use 17 years of monitoring data (from 1993 to 2010) collected in a mountainous region of central Italy (Arezzo, Tuscany) in an ecological niche-based model (MaxEnt) to characterize breeding sites (i.e. the areas where pups were raised) within home ranges, as detected from play-back responses. From a suite of variables related to topography, habitat and human disturbance we found that elevation and distance to protected areas were most important in explaining the locality of wolf responses. Rendezvous sites (family play-back response sites) typically occurred between 800 and 1200 m a.s.l., inside protected areas, and were usually located along mountain chains distant from human settlements and roads. In these areas human disturbance is low and the densities of ungulates are typically high. Over recent years, rendezvous sites have occurred closer to urban areas as the wolf population has continued to expand, despite the consequent human disturbance. This suggests that undisturbed landscapes may be reaching their carrying capacity for wolves. This, in turn, may lead to the potential for increased human-wolf interactions in future. Applying our model, both within and beyond the species' current range, we identify sites both within the current range and also further afield, that the species could occupy in future. Our work underlines the importance of the present protected areas network in facilitating the recolonisation by wolves. Our projections of suitability of sites for future establishment as the population continues to expand could inform planning to minimize future wolf-human conflicts.

Biodistribution studies of polymeric nanoparticles for drug delivery in mice.

Abstract Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.

Growth on poly(L-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells.

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Biodistribution and molecular studies on orally administered nanoparticle-AON complexes encapsulated with alginate aiming at inducing dystrophin rescue in mdx mice.

We have previously demonstrated that intraperitoneal injections of 2'-O-methyl-phosphorothioate (2'OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles of mdx mice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were tracked in vivo as well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, in mdx mice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

Prey selection by an apex predator: the importance of sampling uncertainty.

The impact of predation on prey populations has long been a focus of ecologists, but a firm understanding of the factors influencing prey selection, a key predictor of that impact, remains elusive. High levels of variability observed in prey selection may reflect true differences in the ecology of different communities but might also reflect a failure to deal adequately with uncertainties in the underlying data. Indeed, our review showed that less than 10% of studies of European wolf predation accounted for sampling uncertainty. Here, we relate annual variability in wolf diet to prey availability and examine temporal patterns in prey selection; in particular, we identify how considering uncertainty alters conclusions regarding prey selection.Over nine years, we collected 1,974 wolf scats and conducted drive censuses of ungulates in Alpe di Catenaia, Italy. We bootstrapped scat and census data within years to construct confidence intervals around estimates of prey use, availability and selection. Wolf diet was dominated by boar (61.5 ± 3.90 [SE] % of biomass eaten) and roe deer (33.7 ± 3.61%). Temporal patterns of prey densities revealed that the proportion of roe deer in wolf diet peaked when boar densities were low, not when roe deer densities were highest. Considering only the two dominant prey types, Manly's standardized selection index using all data across years indicated selection for boar (mean = 0.73 ± 0.023). However, sampling error resulted in wide confidence intervals around estimates of prey selection. Thus, despite considerable variation in yearly estimates, confidence intervals for all years overlapped. Failing to consider such uncertainty could lead erroneously to the assumption of differences in prey selection among years. This study highlights the importance of considering temporal variation in relative prey availability and accounting for sampling uncertainty when interpreting the results of dietary studies.

Persistent dystrophin protein restoration 90 days after a course of intraperitoneally administered naked 2'OMePS AON and ZM2 NP-AON complexes in mdx mice.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.

Exon skipping-mediated dystrophin reading frame restoration for small mutations.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.